Clostridium tetani: Properties, Pathogenesis and Laboratory diagnosis

Clostridium tetani is an obligate anaerobic, gram-positive bacillus with terminal round spore (drum stick appearance). It is the causative agent of tetanus, a vaccine-preventable disease that classically follows a puncture wound by a rusty nail (or any object contaminated with spores of C. tetani). Tetanus is manifested by skeletal muscle spasm and autonomic nervous system disturbance. Tetanus only affects skeletal muscle, smooth muscle, and cardiac muscle function normally.

Tetanus has been known since ancient times; however, its causative organism was isolated by Kitasato in 1889.

Pathogenesis

Mode of transmission

C. tetani are primarily saprophytic (lives in the dead or decaying matter) and only incidentally infect humans. There is no person-to-person spread of C. tetani. Human acquires infection when Clostridium tetani endospores are inoculated into sterile body tissues through traumas (for example; lacerations, deep puncture or crush injuries) or fecal contamination of umbilical cord (neonatal tetanus). The organism would grow and produce tetanus toxin in the anoxic zones created by local tissue death.

Virulence Factors

C. tetani produces two exotoxinstetanolysin and tetanospasmin.

  1. Tetanolysin is a heat-labile, oxygen labile hemolysin antigenically related to the oxygen labile hemolysins produced by C. perfringens, S. pyogenes, and S. pneumoniae. Tetanolysin damages otherwise viable tissue surrounding the infection and optimizes the conditions for bacterial multiplication.
  2. Tetanospasmin (or tetanus toxin) is a heat-labile, oxygen stable, plasmid coded neurotoxin responsible for disease manifestations. Tetanus neurotoxin is released upon cell lysis after bacterial growth under anaerobic conditions (e.g., in deep puncture wounds).It is antigenic and is specifically neutralized by its antitoxin. Its toxoid form is used for vaccine preparation.

Mechanism of Tetanus Toxin

Tetanus toxin binds to polysialogangliosides receptors present on motor nerve terminals which results in toxin internalization. Following internalization, tetanus toxin gets transported in a retrograde way from the peripheral nervous system to the central nervous system by retrograde axonal transport. When tetanus toxin reaches inhibitory neuron terminals, it prevents the presynaptic release of inhibitory neurotransmitters glycine and gamma-aminobutyric acid (GABA).

Tetanus toxin mechanism
Route of tetanus toxin, from entry into an α-motor neuron to its site of action in an inhibitory neuron in the central nervous system (CNS).
(Image source: sciencedirect.com)

In normal condition, Glycine and GABA released from inhibitory interneurons induce muscle relaxation by acting on the motor neurons and blocking excitation and release of acetylcholine at the motor endplate.

Lack of inhibitory signals to the motor neurons and constant release of acetylcholine to the muscle fibers leads to irreversible contraction of the muscles and spastic paralysis.

Mechanism of action of tetanus toxin
Mechanism of action of tetanus toxin

Flaccid paralysis vs. Spastic paralysis
Flaccid paralysis occurs when the muscle cannot contract at all. The muscle stays weak and floppy.
Spastic paralysis occurs when the muscle stays in contraction. The muscle is too rigid and the patient can not move the muscle properly.  It causes muscles to twitch uncontrollably or spasm.

Clinical manifestations of Tetanus

Incubation period of tetanus is about 6-10 days. Shorter the incubation period, graver is the prognosis. Muscles of the face and jaw are often affected first (due to shorter distances for the toxin to reach the presynaptic terminals).

Clinical Manifestations and Complications of Tetanus
Clinical Manifestations and Complications of Tetanus
(Image source: osmosis.org)

Patients have prolonged muscle spasms of both flexor and extensor muscles. Patients with tetanus have spastic muscle contractions, difficulty opening the jaw (called lockjaw, “trismus”), a characteristic smile called “risus sardonicus” and contractions of back muscles resulting in backward arching (Opisthotonos position). Patients are extremely irritable, and tetanic seizures develop, brought about by violent, painful muscle contractions following some minor stimulus, such as noise.

Laboratory Diagnosis

The diagnosis of tetanus is clinical and does not require a demonstration of C. tetani. Treatment should be started immediately based on clinical diagnosis. Laboratory diagnosis provides supportive evidence for confirmation.

Specimen

Excised tissue bits from the necrotic depths of wounds are more reliable than wound swabs.

Gram staining

Spores of Clostridium tetani

Gram staining reveals gram-positive bacilli with terminal and round spores (drum stick appearance or tennis racket appearance). However, microscopy alone is unreliable as it cannot distinguish C. tetani from morphologically similar non-pathogenic clostridia like C. tetanomorphum and C. sphenoides.

Culture

Culture is more reliable than microscopy.

  1. Robertson cooked meat(RCM) brothC. tetani being proteolytic turns the meat particles black and produces a foul odor.
  2. Blood agar with polymyxin B: C. tetani produce characteristic swarming growth when incubated at 37°C for 24-48 hours under anaerobic conditions.

Toxigenicity Test

As pathogenesis of tetanus is toxin mediated, the association of the isolated organism can only be established when its toxin production is demonstrated. Toxigenicity can be detected by both in vitro and in vivo methods.

  1. In vitro hemolysis inhibition test: C. tetani produces hemolysis on blood agar which is inhibited by adding antitoxin. This test indicates the production of tetanolysin only but not tetanospasmin.
  2. In vivo mouse inoculation test: RCM broth with black turbid growth is injected into the root of the tail of a test mouse. The test animal develops stiffness which begins with the tail and progresses to involve the hind limbs on the inoculated side- the other limb-trunk-forelimbs. Death occurs within two days. This test indicates the production of tetanospasmin.

References and further readings

About Acharya Tankeshwar 452 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.

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